417,614 research outputs found
Efficacy of MS-275, a selective inhibitor of class I histone deacetylases, in human colon cancer models
N-(2-aminophenyl)-4-[N-(pyridine-3yl-methoxy-carbonyl) aminomethyl] benzamide (MS-275) is a second generation histone deacetylase (HDAC) inhibitor with significant anti-tumor efficacy currently in clinical development. We investigated the effect of MS-275 treatment on various colon cancer cell lines, as well as on mouse xenograft models derived from human colorectal cancer. MS-275 exerted strong anti-proliferative effects in five cell lines and increased the acetylation of histones 3 and 4. In vivo testing of the compound in eight different models of human colon cancer derived from primary colorectal cancers or from established cell lines revealed that five models were responders, two non-responders and one an anti-responder. Gene expression profiles were determined in order to identify genes and pathways differentially regulated upon MS-275 treatment in responder versus non-responder models. Principle component analysis revealed a correlation of the anti-tumor efficacy with the sub-clustering of the MS-275 treatment groups in 7 out of 8 models. Although the overall gene expression pattern was rather unique for each individual model, 129 genes were significantly up- and 58 genes significantly down-regulated in at least 2 out of 5 responder models in response to MS-275 treatment. We identified potential biomarkers for response to MS-275, such as PRA1, MYADM and PALM2-AKAP2 which were up-regulated in all responder models and down-regulated or unchanged in all non-responder models. Our results provide a starting point for the development of clinically relevant biomarkers for predicting a response to MS-275 and the understanding of the mode of action of this HDAC inhibitor
Investigation into the use of histone deacetylase inhibitor MS-275 as a topical agent for the prevention and treatment of cutaneous squamous cell carcinoma in an SKH-1 hairless mouse model
<div><p>Cutaneous squamous cell carcinomas are a common form of highly mutated keratinocyte skin cancers that are of particular concern in immunocompromised patients. Here we report on the efficacy of topically applied MS-275, a clinically used histone deacetylase inhibitor, for the treatment and management of this disease. At 2 mg/kg, MS-275 significantly decreased tumor burden in an SKH-1 hairless mouse model of UVB radiation-induced skin carcinogenesis. MS-275 was cell permeable as a topical formulation and induced histone acetylation changes in mouse tumor tissue. MS-275 was also effective at inhibiting the proliferation of patient derived cutaneous squamous cell carcinoma lines and was particularly potent toward cells isolated from a regional metastasis on an immunocompromised individual. Our findings support the use of alternative routes of administration for histone deacetylase inhibitors in the treatment of high-risk squamous cell carcinoma which may ultimately lead to more precise delivery and reduced systemic toxicity.</p></div
MS-275 (Entinostat) Promotes Radio-sensitivity in PAX3-FOXO1 Rhabdomyosarcoma cells
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. About 25% of RMS expresses fusion oncoproteins such as PAX3/PAX7‐FOXO1 (fusion‐positive, FP) while fusion‐negative (FN)‐RMS harbors RAS mutations. Radiotherapy (RT) plays a crucial role in local
control but metastatic RMS is often radio‐resistant. HDAC inhibitors (HDACi) radio‐sensitize different cancer cells types. Thus, we evaluated MS−275 (Entinostat), a Class I and IV HDACi, in combination with RT on RMS cells in vitro and in vivo. MS−275 reversibly hampered cell survival
in vitro in FN‐RMS RD (RASmut) and irreversibly in FP‐RMS RH30 cell lines down‐regulating cyclin A, B, and D1, up‐regulating p21 and p27 and reducing ERKs activity, and c‐Myc expression in RD and PI3K/Akt/mTOR activity and N‐Myc expression in RH30 cells. Further, MS−275 and RT
combination reduced colony formation ability of RH30 cells. In both cell lines, co‐treatment increased DNA damage repair inhibition and reactive oxygen species formation, down‐regulated NRF2, SOD, CAT and GPx4 anti‐oxidant genes and improved RT ability to induce G2 growth arrest.
MS−275 inhibited in vivo growth of RH30 cells and completely prevented the growth of RT‐unresponsive RH30 xenografts when combined with radiation. Thus, MS−275 could be considered as a radio‐sensitizing agent for the treatment of intrinsically radio‐resistant PAX3‐FOXO1 RMS
MS-275 synergistically enhances the growth inhibitory effects of RAMBA VN/66-1 in hormone-insensitive PC-3 prostate cancer cells and tumours
Combining drugs, which target different signalling pathways, often decreases adverse side effects while increasing the efficacy of treatment. The objective of our study was to determine if the combination of our novel atypical retinoic acid metabolism-blocking agent (RAMBA) VN/66-1 and a promising histone deacetylase inhibitor N-(2-aminophenyl)4-[N-(pyridine-3-yl-methoxy-carbonyl)aminomethyl]benzamide (MS-275) would show enhanced antineoplastic activity on human PC-3 prostate cancer cells/tumours and also to decipher the molecular mechanisms of action. The combination of VN/66-1+MS-275 was found to be synergistic in inhibiting PC-3 cell growth, caused cell cytostaticity/cytotoxicity and induced marked G2/M phase arrest and apoptosis. In mice with well-established PC-3 tumours, VN/66-1 (5 and 10 mg kg−1 day−1) caused significant suppression of tumour growth compared with mice receiving vehicle alone. Furthermore, treatment with VN/66-1 (10 mg kg−1 day−1)+MS-275 (2.5 mg kg−1 day−1) for 18 days resulted in an 85% reduction in final mean tumour volume compared with control and was more effective than either agent alone. Mechanistic studies indicated that treatment of PC-3 cells/tumours with VN/66-1+MS-275 caused DNA damage (upregulation of γH2AX), hyperacetylation of histones H3 and H4, upregulation of retinoic acid receptor-β, p21WAF1/CIP1, E-cadherin, and Bad and downregulation of Bcl-2. These data suggest that the mechanism of action of the combination of agents is DNA damage-induced p21 activation, resulting in inhibition of the Cdc2/cyclin B complex and accumulation of cells in G2/M phase. In addition, the combination caused modulation and induction of apoptosis. These results suggest that VN/66-1 or its combination with MS-275 may be a novel therapy for the treatment of prostate carcinoma
Anti-Leukemia Activity of MS-275 Histone Deacetylase Inhibitor Implicates 4-1BBL/4-1BB Immunomodulatory Functions
Histone deacetylase inhibitors (HDACi) have demonstrated promising therapeutic potential in clinical trials for hematological malignancies. HDACi, such as SAHA/Vorinostat, Trichostatin A, and MS-275 were found to induce apoptosis of leukemic blasts through activation of the death receptor pathway and transcriptional induction of the Tumor Necrosis Factor (TNF)-related pro-apoptotic family members, TRAIL and FasL. The impact of HDACi on TNF-related costimulatory molecules such as 4-1BB ligand (4-1BBL/TNFSF9) is however not known. Following exposure to SAHA/Vorinostat, Trichostatin A, and MS-275, transcript levels were determined by real time PCR in Jurkat, Raji and U937 cells. Treatment with HDACi up-regulated TNFSF9 gene expression in the three leukemia cell lines, yet to different extend and with distinct kinetics, which did not require de novo protein synthesis and was not associated with DNAse I hypersensitive chromatin remodeling. Transcriptional activity of TNFSF9 promoter-luciferase constructs was induced up to 12 fold by HDACi, and implication of Sp1/Sp3 transcription factors binding to functional GC-box elements was evidenced by reporter gene assays, site-directed mutagenesis, and electrophoretic mobility shift assays. Functionality of modulated target genes was assessed in allogeneic mixed leukocyte reaction experiments. MS-275- and to a lesser extent Trichostatin A- and SAHA-treated Raji cells significantly up regulated T lymphocytes proliferation which was reduced by about 50% by a 4-1BB blocking recombinant protein, while MS-275- but neither Trichostatin A- nor SAHA-treated cells up-regulated IFNγ secretion by T lymphocytes. Our results identify 4-1BBL/4-1BB as a downstream target of HDACi, especially of MS-275 anti-leukemia action in vitro. Thus, HDACi such as MS-275 displaying dual TNF-dependent proapoptotic and costimulatory activities might be favored for inclusion in HDACi-based anti-cancer therapeutic strategies
Targeted acetylation of NF-kappaB/RelA and histones by epigenetic drugs reduces post-ischemic brain injury in mice with an extended therapeutic window.
Nuclear factor-kappaB (NF-κB) p50/RelA is a key molecule with a dual effect in the progression of ischemic stroke. In harmful ischemia, but not in preconditioning insult, neurotoxic activation of p50/RelA is characterized by RelA-specific acetylation at Lys310 (K310) and deacetylation at other Lys residues. The derangement of RelA acetylation is associated with activation of Bim promoter. Objective: With the aim of producing neuroprotection by correcting altered acetylation of RelA in brain ischemia, we combined the pharmacological inhibition of histone deacetylase (HDAC) 1-3, the enzymes known to reduce global RelA acetylation, and the activation of sirtuin 1, endowed with a specific deacetylase activity on the K310 residue of RelA. To afford this aim, we tested the clinically used HDAC 1-3 inhibitor entinostat (MS-275) and the sirtuin 1 activator resveratrol. Methods: We used the mouse model of transient middle cerebral artery occlusion (MCAO) and primary cortical neurons exposed to oxygen glucose deprivation (OGD). Results: The combined use of MS-275 and resveratrol, by restoring normal RelA acetylation, elicited a synergistic neuroprotection in neurons exposed to OGD. This effect correlated with MS-275 capability to increase total RelA acetylation and resveratrol capability to reduce RelA K310 acetylation through the activation of an AMP-activated protein kinase-sirtuin 1 pathway. The synergistic treatment reproduced the acetylation state of RelA peculiar of preconditioning ischemia. Neurons exposed to the combined drugs totally recovered the optimal histone H3 acetylation.Neuroprotection was reproduced in mice subjected to MCAO and treated with MS-275 (20μg/kg and 200μg/kg) or resveratrol (6800μg/kg) individually. However, the administration of lowest doses of MS-275 (2μg/kg) and resveratrol (68μg/kg) synergistically reduced infarct volume and neurological deficits. Importantly, the treatment was effective even when administered 7h after the stroke onset. Chromatin immunoprecipitation analysis of cortices harvested from treated mice showed that the RelA binding and histone acetylation increased at the Bcl-x L promoter and decreased at the Bim promoter. Conclusion: Our study reveals that epigenetic therapy shaping acetylation of both RelA and histones may be a promising strategy to limit post-ischemic injury with an extended therapeutic window
Mechanisms of response of Mesothelial Cells to viral infections: role of epigenetic HDAC1/2 regulation
The main focus of this doctoral thesis is the analysis of the response of mesothelial cells (MCs) to viral infections. As a cellular model we used first primary MCs from
patients undergoing peritoneal dialysis (PD). In mammals, the recognition of pathogen infection occurs trough pathogen recognition receptors (PRRs), and among them, Toll-like receptor (TLR) family plays an important role. TLR3 is one of the TLRs associated to the recognition of viral infection and its role in MC plasticity has not been fully clarified yet. Here, we first analysed the TLRs pattern expression in primary MCs from dialyzed patients. We found that they express a specific subset composed by TRL1, TLR2,
TLR3 and TLR5. We then, characterized the effects of TLR3 stimulation with Poly(I:C). We observed changes in MC cellular plasticity indicating the occurrence of bona fide mesothelial to mesenchymal transition (MMT), characterized by the acquisition of a spindle-like morphology, loss of epithelial markers and induction of mesenchymal markers, including the EMT master gene Snail. Moreover, Poly(I:C) stimulation promoted the induction of a pro-inflammatory response as shown by secretion of inflammatory cytokines and chemokines such as IL-1β, TNFα, IL-6, CXCL8 and CXCL10. Epigenetic reprogramming is a potentially relevant mechanism governing these changes in MC cell plasticity. Here, we analysed the impact of histone deacetylase (HDAC) inhibition using MS-275, an HDAC1/2 pharmacological inhibitor. Quantitative mass spectrometry analysis revealed several pathways altered by MS-275 treatment, including mesenchymal genes, actin cytoskeleton, extracellular matrix, and type-I interferon response regulation. Results obtained by proteomic analysis were then validated by Western blot analysis. Thus, we confirmed the role of MS-275 in promoting the expression of epithelial markers and in the downregulation of the IFNβ-driven response in Poly(I:C) stimulated MCs, which was linked to STAT1 tyrosine phosphorylation. To directly analyse the effects of viral infections on MCs, we then evaluated the response of a pleura mesothelial cell line, MeT5A cells, to SARS-CoV-2 infection. First, we found that MCs express the specific receptors/coreceptors ACE2, TMPRSS2, NRP1 and ADAM17. Moreover, MeT5A were found responsive to SARS-CoV-2 infection. MeT5A cells reacted to viral stimulation through a specific cytokine expression profile characterized by the predominance of an anti-inflammatory over a pro-inflammatory phenotype. Next, we evaluated the role of HDAC1/2 inhibition in SARS-CoV-2 infection. Treatment with MS-275 favoured SARS-CoV-2 infection and productive replication in MeT5A cells. We provided two different mechanisms by which HDAC1/2 inhibition can cause viral spreading in MCs. We found that MS-275 treatment both induced ACE2 and TMPPRSS2 expression and impaired interferon type-I response to SARS-CoV-2 infection. Mechanistically, treatment with MS-275 increased the H3 histone acetylation at ACE2 and TMPRSS2 promoter regions. Last, to find direct evidence of COVID-19 infection in mesothelium, we analysed 4 pleural autoptic samples of COVID-19 patients comparing them with 4 non COVID-19 -infected patients. Immunohistochemical analysis of samples from COVID-19 patients demonstrated a specific alteration of pleura characterized by disruption of the MC monolayer and invasion of the sub-mesothelial stroma by spindle-like MCs, but not evidence of direct MC infection by SARS-CoV-2. Overall, in this study we provided a characterization of changes in MC plasticity upon exposure to virus-related stimuli. Moreover, our data raise a concern on the use of class I HDACs pharmacological inhibitors such as MS-275 and derivatives
in immunocompromised patients since they can potentiate SARS-CoV-2 infectivity
Site-specific quantification of lysine acetylation in the N-terminal tail of histone H4 using a double-labelling, targeted UHPLC MS/MS approach
We developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the site-specific quantification of lysine acetylation in the N-terminal region of histone H4 by combining chemical derivatization at the protein and peptide levels with digestion using chymotrypsin and trypsin. Unmodified epsilon-amino groups were first modified with propionic acid anhydride and the derivatized protein digested with trypsin and chymotrypsin. The newly formed peptide N-termini were subjected to a second derivatization step with d(6)- (heavy) or d(0)- (light) acetic acid anhydride. Samples were mixed at different ratios and peptides monitored by multiple reaction monitoring (MRM) LC-MS/MS. The method was validated in terms of linearity (R (2) a parts per thousand yenaEuro parts per thousand 0.94), precision (RSD a parts per thousand currency signaEuro parts per thousand 10 %), and accuracy (a parts per thousand currency sign27 %) and used to assess the effect of the histone deacetylase (HDAC) inhibitors SAHA and MS-275 in the murine macrophage-like cell line RAW 264.7. SAHA and MS-275 showed site-specific effects on the acetylation levels of K5 and K8 with the K5(Ac)-K8 and K5-K8(Ac) peptides increasing 2.5-fold and 5-fold upon treatment with SAHA and MS-275, respectively. Assessing lysine acetylation in a site-specific manner is important for gaining a better understanding of the effects of HDAC inhibitors and for clarifying disease mechanisms where lysine acetylation plays a role
Regulation of ROCK1 via Notch1 during breast cancer cell migration into dense matrices
Background The behaviour of tumour cells depends on factors such as genetics and the tumour microenvironment. The latter plays a crucial role in normal mammary gland development and also in breast cancer initiation and progression. Breast cancer tissues tend to be highly desmoplastic and dense matrix as a pre-existing condition poses one of the highest risk factors for cancer development. However, matrix influence on tumour cell gene expression and behaviour such as cell migration is not fully elucidated. Results We generated high-density (HD) matrices that mimicked tumour collagen content of 20 mg/cm3 that were ~14-fold stiffer than low-density (LD) matrix of 1 mg/cm3. Live-cell imaging showed breast cancer cells utilizing cytoplasmic streaming and cell body contractility for migration within HD matrix. Cell migration was blocked in the presence of both the ROCK inhibitor, Y-27632, and the MMP inhibitor, GM6001, but not by the drugs individually. This suggests roles for ROCK1 and MMP in cell migration are complicated by compensatory mechanisms. ROCK1 expression and protein activity, were significantly upregulated in HD matrix but these were blocked by treatment with a histone deacetylase (HDAC) inhibitor, MS-275. In HD matrix, the inhibition of ROCK1 by MS-275 was indirect and relied upon protein synthesis and Notch1. Inhibition of Notch1 using pooled siRNA or DAPT abrogated the inhibition of ROCK1 by MS-275. Conclusion Increased matrix density elevates ROCK1 activity, which aids in cell migration via cell contractility. The upregulation of ROCK1 is epigenetically regulated in an indirect manner involving the repression of Notch1. This is demonstrated from inhibition of HDACs by MS-275, which caused an upregulation of Notch1 levels leading to blockade of ROCK1 expression
Preclinical evaluation of the antineoplastic action of 5-aza-2'-deoxycytidine and different histone deacetylase inhibitors on human Ewing's sarcoma cells
<p>Abstract</p> <p>Background</p> <p>Most patients with advanced Ewing's sarcoma (EWS) respond poorly to conventional chemotherapy, indicating the need for new treatment approaches. Epigenetic events, such as promoter hypermethylation and chromatin histone deacetylation, silence the expression of tumor suppressor genes (TSGs) and play an important role in tumorigenesis. These epigenetic changes can be reversed by using 5-aza-2'-deoxycytidine (5AZA-CdR), a potent inhibitor of DNA methylation, in combination with an inhibitor of histone deacetylase (HDAC).</p> <p>Results</p> <p>Here, we used a clonogenic assay to evaluate the <it>in vitro </it>antineoplastic activity of 5AZA-CdR in combination with different HDAC inhibitors on EWS cells. We observed that the HDAC inhibitors, MS-275, trichostatin-A, phenylbutyrate, LAQ824 and depsipeptide, enhanced the antineoplastic action of 5AZA-CdR on EWS cells. The combination of 5AZA-CdR and MS-275 showed marked synergy, and was correlated with significant reactivation of the expression of two TSGs, E-cadherin and tumor suppressor lung cancer-1 (TSLC1), in a EWS cell line.</p> <p>Conclusion</p> <p>These results suggest the value of future clinical studies investigating the combination of 5AZA-CdR and MS-275 in patients with advanced EWS.</p
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